The long term goal of this research program is to precisely determine how a family of positive-strand RNA viruses, the coronaviruses, regulate the availability and activity of the proteins involved in viral RNA transcription and replication. The coronavirus mouse hepatitis virus (MHV) expresses an 800 kDa polyprotein from gene 1 of the genome RNA. The MHV gene 1 polyprotein is postulated to include the activities required for viral RNA synthesis as well as proteinase(s) responsible for maturation processing of individual proteins. Processing of the gene 1 polyprotein is thought to be mediated largely by a viral proteinase (3CLpro) that is expressed as part of the polyprotein. The central hypothesis proposes a critical role for 3CLpro in regulation of MHV gene expression. Therefore, the main objective of this proposal is to determine the role of the MHV 3CLprp in the processing of the MHV gene 1 polyprotein. Specific aims are proposed to: 1) define the amino acid determinants of 3CLpro activity; 2) determine the cleavage site specifically of 3CLpro; and 3) define the proteins processed from the polyprotein by 3CLpro in virus-infected cells. These studies will help us to identify other proteins encoded by the MHV gene 1 polyprotein and determine their functions and interactions during virus replication. We also will gain important new insights into the mechanisms that positive-strand RNA viruses use to regulate the proteins involved in RNA replication. The proposal will strengthen and expand Dr. Densison~s research program by focusing on a defined functional domain within the viral polyprotein. The proposed studies also will allow more detailed studies of the structure/function relationships of viral proteinases and their substrates. Dr. Denison~s career development will be enhanced by interaction with new collaborators, aquisition of new research expertise in protein biochemistry, participation in courses in protein structure and function and by the strong interdisciplinary research environment at Vanderbilt.